Industrial processes that use or generate toxic organic compounds has lead to the contamination of nearby water and land. Most approaches to decontamination or "remediation" involve stopping the local dumping of such compounds and transport of the waste to another area for containment. This is costly and does not eliminate the hazard.
As a remediation technology, bioremediation is considerably more attractive. Rather than merely transporting wastes, it offers the possibility of degrading toxic compounds to harmless reaction products by the use of biologicals.
Bioremediation field trials have involved both in-situ and ex-situ treatment methods. Typically, ex-situ treatment involves the transfer of contaminated waste from the site into a treatment tank designed to support microbial growth, i.e., a "bioreactor". The reactor provides for effective mixing of nutrients and control over temperature, pH and aeration to allow optimum microbial growth.
In-situ treatment involves adding biologicals directly to the waste. This avoids the problems associated with handling (e.g., pumping) toxic compounds. However, in-situ treatment has its own problems. Unlike bioreactors, where microbial growth can be monitored and adjusted, in-situ environmental conditions are difficult to measure and control.
Bioremediation technologies being developed to deal with waste differ further in their ability to handle specific types of compounds. For example, the effluents from most pulp or paper-making operations contain lignin or its degradation products. Lignin is an extremely complex polymer, constituting up to 35% of dry wood weight. During pulping processes, cellulose fibers must be liberated from the surrounding lignin matrix so that they can be associated with one another. The resulting free lignin is highly resistant to degradation.
Chang et al. describe the degradation of the complex lignin polymer by direct addition of cultures of the white-rot fungus, P. chrysosporium. See U.S. Pat. No. 4,655,926, hereby incorporated by reference. They describe the induction of lignin degradation in response to carbon and nitrogen starvation.
This fungal lignin metabolism is a secondary metabolic event. Lignin degradation allows the fungus to expose the cellulose contained within the lignin matrix as its primary food source. Indeed, lignin alone will not support P. chrysosporium growth.
Since cellulose fibers are the primary food source, degradation of lignin by adding the fungus directly to the wood results in reduced pulp yield and an inferior pulp product. Farrell has proposed, therefore, an improvement in lignin degradation. See U.S. Pat. Nos. 4,687,745 and 4,690,895, hereby incorporated by reference. She describes the use of fungal enzymes rather than fungal cultures. The enzymes degrade lignin without degrading cellulose fibers.
The mechanism by which these enzymes degrade lignin has been investigated. Glenn et al. have characterized the enzyme manganese peroxidase. See Arch. Biochem. Biophys. 251:688 (1986). By separating the enzyme from the substrate using a membrane, they showed that Mn(III) complexed to lactate or other hydroxy acids are intermediates capable of oxidizing organic compounds. This work was confirmed by Lackner et al., Biochem. Biophys. Res. Comm. 178:1092 (1991).
Lignin is but one by-product of paper making operations. The bleaching of paper with chlorine generates effluents that are a serious health concern. Many of these compounds are known to cause cancer in humans. Most importantly, these compounds are not degraded rapidly in the natural environment.
Chang et al. teaches that chloro-organics contained in liquid waste can also be degraded by the white-rot fungus. See U.S. Pat. No. 4,554,075, hereby incorporated by reference. In the method proposed, the fungus is immersed in the liquid containing chloro-organics and periodically exposed to an oxygen enriched atmosphere. The chloro-organics are converted from aromatics to aliphatics.
Unfortunately, the rate of chloro-organic degradation is slow since the degradation activity must be induced by starvation of the organism. To avoid the necessity for this starvation step, Aust et al. teach the direct addition of fungal enzymes rather than whole organisms. See U.S. Pat. No. 4,891,320, hereby incorporated by reference. They suggest the direct addition of a fungal peroxidase. This, however, requires the continually mixing of the enzyme with hydrogen peroxide. This is difficult whether done in an open environment or a closed reactor.
Indeed, the commercial use of fungal degradation is hampered by complex technical and engineering issues. Growth of the organism and/or enzyme production may be inhibited by waste components. See generally, Mileski et al., Appl. Environ. Microbiol. 54:2885 (1988). Furthermore, the enzyme is a protein and, as such, can undergo proteolysis by any number of proteases in the waste stream, and be rendered thereby inactive. See Dosoretz et al., Appl. Environ. Microbiol. 56:3429 (1990). In sum, the degrading ability of P. chrysosporium is not practically maintained for a very long period of time. See Lin et al., Biotechnology and Bioengineering 35:1125 (1990).
There remains a need to develop a bioremediation procedure that can be operated economically on a commercial scale. Such a procedure must be able to deal with diverse waste streams without significant inhibition of the degradation process.